Microorganisms are the most diverse and plentiful form of life on Earth. Many species of microorganisms are harmful to animals or humans, and for this reason antimicrobial compounds are given to humans for microbial infections, and to livestock animals as regular food additives. Worldwide overuse of antibiotic compounds is a great concern to health-care providers, because of the relatively recent discovery of the evolution of multi-drug-resistant (MDR) organisms. Many of these MDR organisms are pathogenic: they are harmful and possibly fatal to animals or humans. They have evolved from strains of organisms that had been easily treated with common antibiotic drugs. Rapid new methods for the determination of antimicrobial susceptibility are needed to screen MDR organisms for the effectiveness of a range of antimicrobial compounds, to determine proper treatment of infections. Furthermore, the detection of unlawful levels of antibiotics in animal carcasses and other human foodstuffs are needed in order to minimize the overuse of antibiotics and prevent or minimize the evolution of further MDR organisms.
Methods for the detection of a microorganism""s antimicrobial susceptibility are diverse. Most established, commercially-available methods rely on the observation of reproduction (growth) over a relatively long period of time (4 hours to several days) and compare the extents of growth for microorganisms cultivated in the absence and presence of antimicrobial compounds. Effective antimicrobial agents reduce or eliminate growth.
More recently, antibiotic susceptibility testing methods have emerged that are based on the direct visual observation or instrumental measurement of changes in the ability of a microorganism to respire, or breathe, in the presence of an antibiotic or cytotoxic compound.
These methods involve the addition of a special chemical compound or mixture of compounds to the culture of microorganisms (cell culture). After an incubation time, a change in color (light absorption wavelength and/or intensity) or fluorescence (light emission wavelength and/or intensity) is observed, either visually or with an instrument. This change occurs because the microorganism transfers electrons to (reduces) the special chemical compound or mixture of compounds. An early example of this principle is contained in U.S. Pat. No. 4,129,483 (Bochner), in which colorless tetrazolium salts are reduced to colored formazan dyes because of respiration. However, drawbacks include the toxicities and insolubilities of the formazan dyes and their low color intensities (molar absorptivities).
U.S. Pat. No. 5,501,959 (Lancaster et al.) describes an antimicrobial susceptibility test in which the dye resazurin is reduced to resorfurin by microorganisms in the presence of poising agents that control the electrochemical potential of the solution: after time is allowed for the microorganisms to reproduce, the measured rate of resazurin reduction is slower in the presence of antibiotic compounds. Resazurin is deep blue, and nonfluorescent, while the reduction product, resorfurin, is red and highly fluorescent. When effective antibiotic compounds are present, the microorganism does not reproduce to the extent necessary to change the solution color from blue to red. However, in this method, growth of the microorganism is essential to yield the red-colored product, because the microorganisms initially present in the sample are at a very low level and must reproduce in order to be able to generate visual or obvious color changes.
U.S. Pat. No. 5,045,477 (Belly et al.) describes special dye molecules, called xe2x80x9cshiftable detectable speciesxe2x80x9d, and methods for their use, where reduction causes the chemical release of a colored or fluorescent product which is readily measurable by the absorption or emission of light of a particular wavelength.
U.S. Pat. No. 5,792,622 (Botsford) describes a method for the microbiological assay of chemicals, based on the inhibition of dye reduction that results from exposure to chemicals that are toxic to the microorganism. The dyes described in this patent are tetrazolium salts, that produce formazan dyes when they are reduced by the microorganism. Measurement of the extent of reduction is performed by absorption spectrophotometry.
The methods described above all make use of light absorbance or emission by compounds that are reduced by microorganisms as they respire. The methods cited above are described for use in antibiotic susceptibility assays.
Several examples exist in which electrochemical methods have been used to measure the extent of reduction of a reducible compound that is either naturally present, such as molecular oxygen, or specially added to the cell culture sample, such as a mediator, or mediator mixture. These methods are based on either potential measurement (potentiometric, near zero current) or current measurement (amperometric, fixed applied potential) techniques. The reasons for making these measurements, and the purposes of the methods, are diverse.
In one method, described in U.S. Pat. No. 3,506,544 (Silverman et al.), the objective is to quantitate or measure enzymes, where the term enzyme is clarified to mean purified enzyme preparations, cell-free extracts, and whole cells. The method requires a first substrate (the mediator), an enzyme (to be quantitated) and a second substrate, with which the enzyme, extract or cell reacts. The mediator compounds described are organic compounds including methylene blue, 2,6-dichlorophenolindophenol, phenosafranin, and phenazine methosulfate. The method includes the amperometric measurement of currents produced by oxidation of enzyme-reduced mediator, using a 2-electrode or a 3-electrode electrochemical cell. In one example, cell concentrations are determined by this method for yeast cells (Saccharomyces cerevisiae).
U.S. Pat. No. 5,126,034 (Carter et al.) describes a device for the electrochemical quantitation of biological cells. In this device, a filter is used to trap and increase the concentration of microorganisms. Amperometric measurements are made, using p-benzoquinone as a mediator. In one example, the screening of several species of bacteria is reported, and these are Bacillus badius, Bacillus cerius, Bacillus sphaericus, Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens and Salmonella typhimurium. 
U.S. Pat. No. 5,576,481 (Beardwood et al.) describes a method to detect microbial fouling of water and an apparatus to perform the method. In this case, a biofilm caused by microbial fouling grows on an electrode material. The measurement uses linear polarization resistance with an AC method for resistance compensation, in order to measure the corrosion current at the electrode material that is caused by the biofilm. The current is caused by the direct oxidation of the electrode material, and mediators are not used. The corrosion current is converted by calculation into a fouling factor, which is used to determine the extent of water fouling by microorganisms.
U.S. Pat. No. 5,611,900 (Worden et al.) describes devices called microbiosensors that use surface-bound enzymes or microorganisms that are bound to an electrode surface to measure the concentrations of different species that are low molecular weight chemical compounds that the microorganisms are capable of metabolizing, or converting to other chemical compounds. The devices described in this patent all include an amperometric oxygen microelectrode to directly measure dissolved oxygen levels. The surface-bound enzymes or microorganisms must consume oxygen in a dose-dependent manner relating to the concentration of the metabolizable low molecular weight chemical compound, and the purpose of these devices is to measure the concentrations of these metabolizable compounds.
U.S. Pat. No. 5,811,255 (Hunter et al.) describes an apparatus and method for aerobic and anaerobic respiration measurements on microorganisms. The microorganism cultures included are aerobic, denitrifying, sulfate-reducing and/or methanogenic. In this patent, measurement of respiration rates and other parameters of the culture are made using ion-selective electrodes, pH electrodes, oxidation-reduction potential electrodes and an ion chromatograph. The measurements made using electrodes are all potentiometric, and relate a measured voltage to the concentration of a low molecular weight, dissolved chemical compound or to the oxidation-reduction potential of the culture. The purpose of this invention is to provide an apparatus and method to be used to optimize the design of wastewater treatment or bioremediation processes.
None of the preceding electrochemical methods or devices have been used to screen microorganisms for their susceptibility to antimicrobial compounds; however, all include the measurement of microorganism respiration rates.
U.S. Pat. No. 4,209,586 (Noller) describes a method for measuring the effectiveness of growth-inhibiting agents on microorganisms in which a potentiometric measurement is used to measure the oxidation-reduction potential of the microorganism culture over a period of time. The normal change in potential, measured without growth-inhibiting agents present, is approximately constant and moves towards more positive values, but if a growth inhibitor is present, the potential moves towards more negative values. This invention relies on measurement of the potential of the culture medium as the cells grow, and does not measure respiration rates.
U.S. Pat. No. 5,348,862 (Pasero et al.) describes a method for the quantitation of microorganisms in liquids. This method employs a filter to capture and increase the quantity of microorganisms in the sample to be tested, and uses amperometric measurement to quantitate the respiration rate of the sample. The measured current is related to the concentration of microorganisms in the sample. In this example, a mediator is used to transport electrons from the microorganisms to the working electrode to generate the respiration-induced current. Typical mediators used in this method are sodium or potassium ferricyanide or benzoquinone or mixtures thereof. The method consists of a first current measurement in the presence of the filter with bacteria, a second step consisting of rinsing the measurement cell and treating the filter with a biocidal liquid to kill bacteria, and a third step consisting of a second current measurement in the presence of the treated filter. Current values observed in the third step are subtracted from current values observed in the first step, and the difference is used to measure the concentration of microorganisms. The biocidal liquids described are solutions of sodium hypochlorite, formaldehyde and chlorine dioxide.
U.S. Pat. No. 5,654,165 (Kusunoki et al.) describes an apparatus and method for screening antibacterial drugs against aerobic microorganisms that normally consume oxygen as they respire. In this case, a test microorganism is added to a solution that contains the antibacterial drug and one that does not contain the drug. Sensitivity of the microorganism to the drug is determined by measuring the resulting concentration of dissolved oxygen in the two solutions using two amperometric oxygen electrodes. Sensitivity to the antibacterial drug is indicated if the two amperometric oxygen electrodes measure different concentrations of dissolved oxygen. Two strains of Escherichia coli as well as Staphylococcus aureus, Kliebsiella pneumoniae and Pseudomonas aeruginosa were used as examples, and the antibiotic drugs that were screened were ampicillin, streptomycin, chloramphenicol, tetracycline and sulfanilamide. This apparatus and method are restricted to the screening of aerobic microorganisms, because oxygen consumption is measured.
This invention provides a rapid new method to be used either for the determination of antibiotic or cytotoxic susceptibility in microorganisms, or for the determination of the presence or absence of antibiotic or cytotoxic compounds in samples using microorganisms with known susceptibilities.
The method for assessing susceptibility of a microorganism to an antibiotic or cytotoxic drug comprises adding a suitable mediator or mediator mixture to a sample of the microorganism in the presence of the drug, and assessing variation of the microorganism""s respiration rate overtime by electrochemical measurement of mediator consumption resulting from microorganism respiration. This is compared with variation of the respiration rate of another sample of the microorganism not exposed to the drug.
The preferred method further comprises a sample preparation step, in which the cell culture or suspension of microorganisms is combined with a solution of the proposed antibiotic or cytotoxic drug and incubated for a fixed time, a second step in which a mediator or mediator mixture is added, and a third step in which an amperometric or coulometric measurement is made at fixed applied potential, using standard, commercially available electrochemical instrumentation (a potentiostat) and either a 2-electrode or a 3-electrode electrochemical cell. The first two steps can be combined into one step in some embodiments. In the absence of antimicrobial compounds, the mediator is converted by the microorganism from the oxidized to the reduced form at a rate that is characteristic of the organism and the concentrations of organism and mediator in the sample. During the measurement step, the reduced mediator is reconverted to the oxidized form at the working electrode by applying a fixed voltage to the working electrode, relative to a reference or counter electrode, and the magnitude of the measured current is proportional to reduced mediator concentration in the sample. When effective antimicrobial compounds are present during the incubation and measurement steps, the rate of mediator reduction, and the resulting measured signals, are significantly different and are usually much lower than the rate measured in the absence of the antimicrobial compound.
The preferred method for the determination the presence or absence of antibiotic or cytotoxic compounds in a sample comprises adding to the sample a microorganism with known susceptibilities and a suitable mediator or mediator mixture, and assessing variation of the microorganism""s respiration rate over time by electrochemical measurement of mediator consumption resulting from microorganism respiration. This rate is compared with variation of the respiration rate of another sample not exposed to such compounds.
The mediator or mediator mixture can be any suitable oxidant, including for example one or more of the following:
ferricyanide (hexacyanoferrate (III) is another name for this);
dichlorophenol-indophenol (DCIP);
ferrocene and ferrocene derivatives;
methylene blue;
janus green;
tris(bipyridyl)iron (III);
the quinone class which includes benzoquinone, naphthoquinone, menadione, anthraquinone, and substituted derivatives of these; and
the phenazine class which includes phenazine methosulfate and phenazine ethosulfate.